Infection of Mammalian Cells by Adenoviral Vectors.
Infection of mammalian cells with adenoviral vectors involves the following phases:                a. binding        b. entry        c. uncoating        d. expression of viral genes        e. replication of DNA        f. expression of viral coat and core proteins        g. assembly of viral particles        h. cell lysis.        
Replication of Adenoviral Vectors and Oncolytic Vectors.
Once the adenovirus enters a mammalian cell, the viral coat proteins unfold, releasing the viral DNA which is normally bound to a viral protein which transports the viral DNA from the cytoplasm across the nuclear membrane into the nucleus of the cell. The genes of the viral DNA are then read out into mRNA in a pre-specified fashion from viral genes which encode proteins for replication of the viral DNA and viral coat proteins. This RNA is translated into replication proteins and coat proteins. This leads to spontaneous assembly of infectious particles with a protein shell surrounding viral DNA bound to transport proteins and polymerases. The production of many infectious particles eventually results in lysis of the cell, leading to cellular death and release of thousands of infectious viral particles. With naturally occurring DNA viruses, this cycle of infection, replication and lysis of the infected cell producing infectious particles occurs in all of the cells of the body (normal cells and tumor cells).
In contrast to naturally occurring viruses, many of the “oncolytic viruses” have been genetically modified such that their replication processes occurs only in cancer cells, not in the normal cells of the body. Thus, these genetically modified viruses can infect tumor cells eventually leading to tumor cell death and the release of many infectious viral particles which have been produced inside the infected tumor cells which reside inside the tumor nodules. This process can continue until the majority of the tumor cells are destroyed.
Such vectors have been under study for several years. The strength of these vectors is that their replication machinery is genetically engineered to be specifically active in the intracellular environment of the cancer cells by making the replication of the vectors dependent on signals only present in the tumor cell. This makes the toxicity of the oncolytic virus specific for the tumor cells, thereby sparing the normal tissues of the body. This is unlike chemotherapy which damages and kills both normal and neoplastic cells.
Limitations of Oncolytic Adenoviral Vectors.
Oncolytic adenoviral vectors have the following features which limit their utility:
1. By making the replication of oncolytic vectors dependent on the regulatory environment of the tumor cells, the level of replication and total tumor cell kill is diminished.
2. Although a potent replication competent oncolytic viral vector can reduce the total body tumor burden significantly, only a fixed fraction of the total number of tumor cells die. Thus, the initial regression of the tumor cell masses which occurs immediately following chemotherapy or administration of an oncolytic vector is followed by regrowth following completion of the therapy.3. The oncolytic adenoviral vector is not antigen specific unless specifically modified for that purpose. The invention outlined in this patent application overcomes these hurdles.4. The oncolytic viruses, like chemotherapy and most forms of cancer treatment, kill only a fraction of the tumor cells, never 100%. This is called the fractional cell kill. The fraction or percentage of the tumor cells which are killed is a property of the type of treatment used, and the features of the particular tumor population which make a portion of the tumor cells resistant to each therapy.5. Tumor cell populations are heterogeneous, and are composed of cells which are resistant and cells which are sensitive to each type of therapy. Following the administration of a therapy to which the majority of tumor cells are sensitive, the tumor nodule shrinks, due to death of the sensitive cells, and then regrows due to the growth of the surviving cancer cells which were resistant to therapy.
The addition of the immunotherapeutic vaccine described in the next section to the Ad-LPE1ACDA virus converts the fractional cell kill into killing of 100% of the tumor cells This conversion to a hundred percent tumor cell kill by the vaccine is conditional upon the number of tumor cells being reduced to a small number by therapies like oncolytic viruses or chemotherapy. These latter types of therapies can tackle larger number of cells than normally is the case for vaccines. As will be outlined below, for an oncolytic vector such as Ad-LPE1ACDA which also carries a TAA/ecdCD40L vaccine transcription unit (which encodes a TAA fused to the ecdCD40L) to successfully eradicate all of the tumor cells in a population, the E1ACDA transcription unit must be tumor specific and the vaccine TAA/ecdCD40L transcription unit must very strong and not necessarily tumor specific other than through the choice of the TAA to link to the ecdCD40L. That is why the TAA/ecdCD40L is driven by a CMV transcriptional promoter, and the E1ACDA transcription unit is driven by a LP promoter.
Historical Summary of the Development by Applicant of the TAA/ecdCD40L Vaccine Platform.
The TAA/ecdCD40L vaccine is based on the attachment of a fragment of a target associated antigen (TAA) fused to the extracellular domain (ecd) of the potent immunostimulatory signal CD40 ligand (CD40L). The vaccine can be administered either as a TAA/ecdCD40L protein, or as an expression vector encoding the TAA/ecdCD40L such as the adenoviral vector (Ad-sig-TAA/ecdCD40L vector), or other viral vectors, or a plasmid DNA expression vector encoding the TAA/ecdCD40L protein (1-11). The vaccine can be also administered as a vector prime followed in 7 and 21 days with sc injections of the TAA/ecdCD40L protein vaccine. This vaccine platform was developed by Applicant's laboratory (1-11) to overcome the following problems: weak immunogenicity of the target antigens, qualitative or quantitative defects of CD4 helper T cells, defective response in immunodeficient individuals including the older aged population due to diminished expression of CD40L in activated CD4 helper T cells, and/or low levels of presentation of target antigens on Class I or II MHC in dendritic cells (DCs). The CD40L is important for the expansion of antigen specific CD8 effector T cells and antigen specific B cells in response to vaccination.
Modes of Administration of TAA/ecdCD40L Vaccine.
There are four versions of this vaccine: 1. One in which the TAA/ecdCD40L transcription unit is embedded in a replication incompetent adenoviral vector (Ad-sig-TAA/ecdCD40L); 2. One in which the expression vector is used as an initial priming injection, followed by two sc injections of the TAA/ecdCD40L protein; 3. One in which the vaccine consists solely of the TAA/ecdCD40L protein; and 4. One in which the TAA/ecdCD40L is inserted into a plasmid DNA expression vector. The TAA is connected through a linker to the aminoterminal end of the ecd of the potent immunostimulatory signal CD40L (1, 3 and 5).
Impact of Attachment of TAA to CD40L.
The attachment of fragments of the TAA to the CD40L accomplishes two things: 1. the binding of the TAA/ecdCD40L protein to the CD40 receptor on the DCs as well as on the B cells and T cells activates these cells thereby promoting a potent immune response (1, 3, 5); 2. once the TAA/ecdCD40L protein is engaged on the CD40 receptor of the DC, the entire TAA/ecdCD40L protein is internalized into the DC in a way that allows Class I as well as Class II MHC presentation of the TAA (1, 5).
Activation of DCs by TAA/ecdCD40L Vaccine.
The activated TAA loaded DCs then migrate to the regional lymph nodes (1, 5) where they can activate and induce expansion of the TAA specific CD8+ effector T cells. These antigen specific CD8+ effector cells become increased in number in the lymph nodes (1, 5), and they then egress from the lymph nodes into the peripheral blood. The antigen specific CD8 effector T cells exit the intravascular compartment and enter into the extra-vascular sites of inflammation or infection (5, 8, 9, and 11). In addition to showing that this vaccine increases the levels of the antigen specific CD8+ effector T cells in the sites of inflammation or infection (11 and 12), the Applicant's laboratory has shown that the activation and expansion of the B cells by the TAA/ecdCD40L protein increases the levels of the TAA specific antibodies (including neutralizing antibodies against viral antigens) in the serum (5, 8, 9, 11 and 12). Vaccines have been described that include an adenoviral expression vector encoding a fusion protein that includes a target associated antigen (TAA) fused to CD40 ligand (CD40L). See, e.g., U.S. Patent Application Publication US 2005-0226888 (application Ser. No. 11/009,533) titled “Methods for Generating Immunity to Antigen,” filed Dec. 10, 2004.
Historical Summary of Vectors which Kill Cancer Cells Through Releasing Chemotherapy in Tumor Cells.
The Applicant's laboratory introduced a 5 fluorocytosine (5-FC) prodrug activation transcriptional unit encoding the cytosine deaminase (CDA) gene driven by the L-plastin (LP) tumor specific promoter into an adenoviral vector (2, 12-14). This vector is called Ad-LPCDA. The CDA gene catalyzes the conversion of a non-toxic prodrug, 5-FC, into the chemotherapy agent 5-fluorouracil (5-FU). They showed that the administration of the Ad-LPCDA vector to mice following intraperitoneal injection of 5-FC to test mice carrying subcutaneous deposits of tumor nodules derived from established tumor cell lines, reduced the growth of these tumor cell lines (12-14).
Applicant's laboratory had also shown that the cytotoxic effect of the Ad-LPE1ACDA vector carrying both the E1A and the CDA transgenes under the influence of the LP promoter, was greater than the vector which contains only the CDA gene prodrug activation transcription unit without the E1A gene, both in an in vitro cell line experiment as well as in an in vivo experiment in human tumor xenograft models (14). The addition of the E1A gene to the CDA gene in the LP driven transcription unit creates a chemotherapy targeting vector which is replication competent only in the tumor cells.
Applicant's laboratory then compared the action of combination chemotherapy for colon cancer with and without the administration of the Ad-LPCDA chemotherapy targeting vector following the intraperioneal injection of 5-FC (2).
The goal of creating a replication competent viral vectors is the direct killing of the target tumor cells by oncolytic effect of the virus (15-16). The goal of using the L-plastin tumor specific promoter to drive the expression of the E1A gene as well as the prodrug activation transcription unit was to increase the therapeutic effect over that seen with replication competent vectors alone or with chemotherapy targeting vectors alone. The resulting vector (Ad-LPE1ACDA) increased the antitumor effect seen with the Ad-LPCDA vector without increasing toxicity to the normal cells (12-14).
Prodrug Activation Transcription Unit Gene Therapy.
Previous reports have shown the tumor suppressive effect of adenoviral vectors carrying the CDA cDNA when used in combination with prodrug 5-FC on various tumor cell lines and in vivo models (12-18). The infectivity of normal as well as tumor cells by the adenoviral vector has represented a disadvantage for these adenoviral vectors since the expression of the therapeutic transgenes in the normal cells generates toxic side effects. In order to overcome this limitation, many groups have been focusing on tumor or tissue specific gene promoters to reduce side effects.
The L-Plastin Tumor Specific Promoter.
The plastins are a family of actin binding proteins which are responsible for functions such as cell division, intracellular trafficking, cell morphology and cell motility. There are three types: L-plastin which is found in hematopoietic cells, T-plastin, which is found in the cells of solid tissues, like in the neurons of the brain, and I-plastin, which is found in the gastrointestinal track and the kidney. L-plastin is not expressed in normal tissues (except for the leukocyte) and is expressed in all epithelial neoplastic cells. The promoter of the L-plastin gene has been used to drive expression of the cytosine deaminase gene in cancer cells (12-14). There are many other “tumor specific” transcriptional promoters (15-16).
The Cytosine Deaminase Prodrug Activation Gene.
Cytosine deaminase (CDA) is a gene found in yeast and bacterial cells, which converts the non-toxic pro-drug, 5-fluorocytosine (5-FC), into a toxic chemotherapy agent, 5-fluorouracil (5-FU). This action is known as a “prodrug activation” gene, in that it converts a non-toxic and inactive chemical, into a fully toxic and active chemical for cancer treatment. There are many other prodrug activation genes and proteins.
Use of the CDA Gene and the L-Plastin Promoter in a Replication Incompetent Adenovirus to Delivery Chemotherapy to Tumor Cells.
The Applicant's laboratory has reported experiments involving vectors carrying the tumor specific L-plastin driven genes (2, 12-14). These results showed that the replication competent viral vector can kill the tumor cell directly by oncolytic effect of the virus. The L-plastin driven CDA or CDA/E1A vectors are selectively toxic to the tumor cell lines without being toxic to the normal cells (12-14).